Methods of vaccine administration

ABSTRACT

This invention relates to a method of treating a dog for canine diseases comprising administering to the dog therapeutically effective amounts of a vaccine, wherein the vaccine comprises viral antigens, a bacterin, or both, and wherein the vaccine is administered subcutaneously or orally according to the schedules provided herein.

FIELD OF INVENTION

This invention relates to a method of treating a dog for canine diseasescomprising administering to the dog therapeutically effective amounts ofvaccine, wherein the vaccine comprises viral antigens, a bacterin, orboth, and wherein the vaccine is administered subcutaneously or orallyaccording to the schedules provided herein.

BACKGROUND OF THE INVENTION

Vaccines against major canine infectious disease have been available forthree to four decades, and they have greatly reduced the incidence ofthese infectious diseases in dogs (Appel, M J. 1999. Adv Vet Med.41:309-324). Pfizer Animal Health sells several vaccines for preventionof diseases associated with various viral and bacterial diseases indogs. The VANGUARD® line of vaccines, including core canine vaccines, isused for the vaccination of healthy dogs 6 weeks of age or older as anaid in preventing canine distemper (CD) caused by canine distemper {CD)virus, infectious canine hepatitis (ICH) caused by canine adenovirustype 1 (CAV-1), respiratory disease caused by canine adenovirus type 2(CAV-2), canine parainfluenza (CPI) caused by canine parainfluenza (CPI)virus, and canine parvoviral enteritis caused by canine parvovirus (CPV)(Mouzin D E, et al., 2004, JAVMA, 224: 55-60).

CD is a high morbidity, high mortality viral disease occurring inunvaccinated dog populations worldwide. Approximately 50% ofnon-vaccinated, non-immune dogs infected with CDV develop clinical signsand approximately 90% of those dogs die (Swango L J. 1983. Norden News58:4-10). ICH, caused by CAV-1, is a universal, sometimes fatal, viraldisease of dogs characterized by hepatic and generalized endotheliallesions. The respiratory disease caused by CAV-2, in severe cases, mayinclude pneumonia and bronchopneumonia. CAV-2 vaccine has been shown tocross-protect against ICH caused by CAV-1 (Bass E P, et al., 1980,JAVMA, 177:234-242). The upper respiratory disease caused by CPI virusmay be mild or subclinical, with signs becoming more severe ifconcurrent infection with other respiratory pathogens exists. Theenteric disease caused by CPV is characterized by sudden onset ofvomiting and diarrhea, often hemorrhagic, and may be accompanied byleukopenia (Appel M J, et al., 1979, Vet Rec, 105:156-159).

Major animal health companies market core canine vaccines in theircompanion animal vaccine franchise. However, all of these vaccines aredelivered by the parenteral route, especially subcutaneous injections. Acanine vaccine that could be delivered easily would provide increasedconvenience of vaccine delivery to the pet, the veterinarian, and thepet owner, and allow for personnel untrained in parenteraladministration techniques to deliver canine core vaccines to animals.

SUMMARY OF THE INVENTION

Provided herein is a method of treating a dog for canine diseasescomprising administering to the dog therapeutically effective amounts ofvaccine, wherein the vaccine comprises viral antigens, a bacterin, orboth, and wherein the vaccine is administered subcutaneously or orallyin a first dose, orally in a second dose, orally in an optional thirddose, and orally in one or more annual doses. The viral antigenscomprise one or more of 1) canine distemper (CD) virus, 2) canineadenovirus type 2 (CAV-2), 3) canine parainfluenza (CPI) virus, 4)canine parvovirus (CPV), 5) and canine coronavirus (CCV), and whereinthe bacterin comprises one or more of Leptospira canicola, L.grippotyphosa, L. icterohaemorrhagiae, L. Pomona, L. bratislava, andBordetella bronchiseptica; and any combination of viral antigens andbacterin thereof.

Also provided herein is a method of treating a dog for canine diseasescomprising administering to the dog therapeutically effective amounts ofvaccine, wherein the vaccine comprises viral antigens, a bacterin, orboth, and wherein the vaccine is administered subcutaneously in a firstand in a second dose, and orally in a third dose, and orally in one ormore annual doses. The viral antigens comprise one or more of 1) CDvirus, 2) CAV-2, 3) CPI virus, 4) CPV, 5) and CCV, and the bacterincomprises one or more of Leptospira canicola, L. grippotyphosa, L.icterohaemorrhagiae, L. Pomona, L. bratislava, and Bordetellabronchiseptica; and any combination of viral antigens and bacterinthereof.

In one embodiment, the vaccine comprises one or more live viruses, oneor more modified-live viruses, one or more inactivated viruses, orcombinations thereof.

In one embodiment, the viral antigens are CD virus, CAV-2, CPI virus,and CPV. In another embodiment, these four viral antigens are combinedwith a bacterin composed of Leptospira canicola, L. grippotyphosa, L.icterohaemorrhagiae, and L. pomona. In yet another embodiment, thesefour viral antigens are combined with a bacterin composed of Bordetellabronchiseptica. In still another embodiment, these four viral antigensare combined with the CCV antigen and a bacterin composed of Leptospiracanicola, L. grippotyphosa, L. icterohaemorrhagiae, and L. pomona.

The treated canine diseases comprise one or more of the following: 1) CDcaused by CD virus; 2) infectious canine hepatitis caused by CAV-1; 3)respiratory disease caused by CAV-2 or respiratory CCV; 4) CPI caused byCPI virus; 5) enteritis caused by CCV or CPV; 6) leptospirosis caused byLeptospira canicola, L. grippotyphosa, L. icterohaemorrhagiae, L.pomona, or L. Bratislava; and 7) infectious tracheobronchitis (“kennelcough”) caused by Bordetella bronchiseptica.

In one embodiment, the treated diseases comprise 1) CD caused by CDvirus; 2) infectious canine hepatitis caused by CAV-1; 3) respiratorydisease caused by CAV-2; 4) CPI caused by CPI virus; 5) and canineparvoviral enteritis caused by CPV.

In one embodiment, the second dose is administered from about 7 to about35 days, inclusive, after the first dose. In a more specific embodiment,the second dose is administered about 3 weeks after the first dose. Inanother embodiment, the third dose is administered from about 7 to about35 days, inclusive, after the second dose. In a more specificembodiment, the third dose is administered about 3 weeks after thesecond dose. In still another embodiment, the annual dose isadministered about one year after the first dose. In yet anotherembodiment, annual doses administered after said annual dose areadministered repeatedly about one year after the immediately priorannual dose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Serological Response (SN) to CDV (mean titers). Animals intreatment groups T02 and T03 were given experimental vaccines firstsubcutaneously (on day 0), then the same vaccine orally three weeks (day21) and six weeks (day 42) later. The animals in group T02 received arelatively low dose, and the animals in group T03 received a relativelyhigh dose. Blood samples were taken weekly to measure concentrations(e.g., titers) of CDV.

FIG. 2 Serological Response (SN) to CPV (mean titers). Animals weretreated as described for FIG. 1. Blood samples were taken weekly tomeasure concentrations (e.g., titers) of CPV.

FIG. 3. Serological Response (SN) to CAV-1 (mean titers). Animals weretreated as described for FIG. 1. Blood samples were taken weekly tomeasure concentrations (e.g., titers) of CAV-1.

DETAILED DESCRIPTION OF THE INVENTION Definitions and Abbreviations

“About” or “approximately,” when used in connection with a measurablenumerical variable, refers to the indicated value of the variable and toall values of the variable that are within the experimental error of theindicated value (e.g., within the 95% confidence interval for the mean)or within 10 percent of the indicated value, whichever is greater,unless about is used in reference to time intervals in weeks where“about 3 weeks,” is 17 to 25 days, and about 2 to about 4 weeks is 10 to40 days.

“Adjuvant” means a pharmaceutically acceptable substance or compositionthat increases the immune response to an antigen.

“Antibody” refers to an immunoglobulin molecule that can bind to aspecific antigen as the result of an immune response to that antigen.Immunoglobulins are serum proteins composed of “light” and “heavy”polypeptide chains having “constant” and “variable” regions and aredivided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on thecomposition of the constant regions.

“Antigen” or “immunogen” refers to a molecule that contains one or moreepitopes (linear, conformational or both) that upon exposure to asubject will induce an immune response that is specific for thatantigen. An epitope is the specific site of the antigen which binds to aT-cell receptor or specific antibody, and typically comprises about 3amino acid residues to about 20 amino acid residues. The term antigenrefers to killed, attenuated or inactivated bacteria or viruses. Theterm antigen also refers to antibodies, such as anti-idiotype antibodiesor fragments thereof, and to synthetic peptide mimotopes that can mimican antigen or antigenic determinant (epitope).

“Excipient” refers to any component of a vaccine that is not an antigen.

“Dose” refers to a vaccine or immunogenic composition given to asubject. A “first dose” or “priming vaccine” refers to the dose of sucha composition given on Day 0. A “second-dose” or a “third dose” or an“annual dose” refers to an amount of such composition given subsequentto the first dose, which may or-may not be the same vaccine orimmunogenic composition as the first dose.

“Immune response” in a subject refers to the development of a humoralimmune response, a cellular immune-response, or a humoral and a cellularimmune response to an antigen. A “humoral immune response” refers to onethat is mediated by antibodies. A “cellular immune response” is onemediated by T-lymphocytes or other white blood cells or both, andincludes the production of cytokines, chemokines and similar moleculesproduced by activated T-cells, white blood cells, or both. Immuneresponses can be determined using standard immunoassays andneutralization assays, which are known in the art.

“Immunologically protective amount” or “effective amount to produce animmune response” of an antigen is an amount effective to induce animmunogenic response in the recipient. The immunogenic response may besufficient for diagnostic purposes or other testing, or may be adequateto prevent signs or symptoms of disease, including adverse healtheffects or complications thereof, caused by infection with a diseaseagent. Either humoral immunity or cell-mediated immunity or both may beinduced. The immunogenic response of an animal to an immunogeniccomposition may be evaluated, e.g., indirectly through measurement ofantibody titers, lymphocyte proliferation assays, or directly throughmonitoring signs and symptoms after challenge with wild type strain,whereas the protective immunity conferred by a vaccine can be evaluatedby measuring, e.g., reduction in clinical signs such as mortality,morbidity, temperature number, overall physical condition, and overallhealth and performance of the subject. The immune response may comprise,without limitation, induction of cellular and/or humoral immunity.

“Immunogenic” means evoking an immune response or antigenic. Thus animmunogenic composition would be any composition that induces an immuneresponse.

“Intranasal” means within the nose. Thus, intranasal administrationrefers to the introduction of a substance, such as a vaccine, into asubject's body through or by way of the nose, as would occur, forexample, by placing one or more droplets in the nose. It involvestransport of the substance primarily through the nasal andnasopharyngeal mucosa.

“N days,” “N” interval or period of time, or “M days” following an eventrefers, respectively, to any time on the N th or M th day after theevent. For example, vaccine a subject with a second vaccine 21 daysfollowing administration of a first vaccine means that the secondvaccine is administered at any time on the 21^(st) day after the firstvaccine. This description is often applied to the interval between afirst and second vaccination, or between a second and third vaccination.

“Oral” or “peroral” administration refers to the introduction of asubstance, such as a vaccine, into a subject's body through or by way ofthe mouth and involves swallowing or transport through the oral mucosa(e.g., sublingual or buccal absorption) or both.

“Oronasal” administration refers to the introduction of a substance,such as a vaccine, into a subject's body through or by way of the noseand the mouth, as would occur, for example, by placing one or moredroplets in the nose. Oronasal administration involves transportprocesses associated with oral and intranasal administration.

“Parenteral administration” refers to the introduction of a substance,such as a vaccine, into a subject's body through or by way of a routethat does not include the digestive tract. Parenteral administrationincludes subcutaneous administration, intramuscular administration,transcutaneous administration, intradermal administration,intraperitoneal administration, intraocular administration, andintravenous administration. For the purposes of this disclosure,parenteral administration excludes administration routes that primarilyinvolve transport of the substance through mucosal tissue in the mouth,nose, trachea, and lungs.

“Pharmaceutically acceptable” refers to substances, which are within thescope of sound medical judgment, suitable for use in contact with thetissues of subjects without undue toxicity, irritation, allergicresponse, and the like, commensurate with a reasonablebenefit-to-risk-ratio, and-effective for their intended use.

“Subject” refers to a canine.

“TCID₅₀” refers to “tissue culture infective dose” and is defined asthat dilution of a virus required to infect 50% of a given batch ofinoculated cell cultures. Various methods known to one skilled in theart may be used to calculate TCID₅₀, including the Spearman-Karbermethod which is utilized throughout this specification. For adescription of the Spearman-Karber method, see B. W. Mahy & H. O.Kangro, Virology Methods Manual 25-46 (1996).

“Therapeutically effective amount,” in the context of this disclosure,refers to an amount of an antigen or vaccine that would induce an immuneresponse in a subject receiving the antigen or vaccine which is adequateto prevent signs or symptoms of disease, including adverse healtheffects or complications thereof, caused by infection with a pathogen,such as a virus or a bacterium. Humoral immunity or cell-mediatedimmunity or both humoral and cell mediated immunity may be induced. Theimmunogenic response of an animal to a vaccine may be evaluated, e.g.,indirectly through measurement of antibody titers, lymphocyteproliferation assays, or directly through monitoring signs and symptomsafter challenge with wild type strain. The protective immunity conferredby a vaccine can be evaluated by measuring, e.g., reduction in clinicalsigns such as mortality, morbidity, temperature number, overall physicalcondition, and overall health and performance of the subject. The amountof a vaccine that is therapeutically effective may vary depending on theparticular virus used, or the condition of the subject, and can bedetermined by a veterinary physician.

“Treating” refers to preventing a disorder, condition, or disease towhich such term applies; or to preventing one or more symptoms of suchdisorder, condition, or disease; or to reversing, alleviating, orinhibiting the progress of such disorder, condition, or disease.

“Treatment” refers to the act of “treating” as defined above.

“Vaccine” refers to a composition that includes an antigen, as definedherein. Administration of the vaccine to a subject results in an immuneresponse, generally against one or more specific diseases. The amount ofa vaccine that is therapeutically effective may vary depending on theparticular virus used, or the condition of the dog, and can bedetermined by a veterinary physician. The vaccine may be introduceddirectly into the subject by the subcutaneous, oral, oronasal, orintranasal routes of administration.

“VANGUARD® Plus 5” is a commercially available vaccine which containscanine distemper (CD) virus, canine adenovirus type 2 (CAV-2), canineparainfluenza (CPI) virus, and canine parvovirus (CPV). “VANGUARD® Plus5 L4” is a commercially available vaccine which contains CD virus,CAV-2, CPI virus, CPV, and Leptospira canicola, L. grippotyphosa, L.icterohaemorrhagiae, and L. pomona. “VANGUARD® Plus 5 L4 CV” is acommercially available vaccine which contains CD virus, CAV-2, CPIvirus, CPV, canine coronavirus (CCV), and Leptospira canicola, L.grippotyphosa, L. icterohaemorrhagiae, and L. pomona. The name VANGUARD®is registered and owned by Pfizer, Inc.

DESCRIPTION OF THE INVENTION

Provided herein is a vaccination regimen that reduces morbidity andmortality in animals, and provides increased ease of administration ofvaccines for pets, veterinarians, and animal owners. It is highlydesirable to administer vaccines using methods that do not requireinjection when animals are older. Also, oral vaccines are probably lesslikely to induce undesirable side effects that may be seen afterparenteral vaccinations. Oral vaccines can be administered byindividuals untrained in adminstering materials parenterally (i.e., viaa needle and syringe), thereby increasing the probable frequency ofvaccination and protection from disease.

The invention provides methods of vaccinating a dog with vaccine orimmunogenic composition as an aid in preventing or treating diseasecaused by virulent viral or bacterial strains, including but not limitedto 1) canine distemper (CD) caused by canine distemper (CD) virus; 2)infectious canine hepatitis (ICH) caused by canine adenovirus type 1(CAV-1); 3) respiratory disease caused by canine adenovirus type 2(CAV-2); 4) canine parainfluenza caused by canine parainfluenza (CPI)virus, and 5) canine parvoviral enteritis caused by canine parvovirus(CPV). Other canine diseases as noted below may also be treated by thisinvention.

The methods comprise administering to the dog therapeutically effectiveamounts of a first dose, a second dose, and optionally, a third dose ofa composition that is capable of inducing an immune response in thesubject. Administration is also given about a year after the first doseas an annual booster. The first dose may be administered subcutaneouslyor orally. The second dose is administered orally about N days followingadministration of the first vaccine. Here, N is an integer from about 7to about 35, inclusive, but is typically an integer from about 14 toabout 28, inclusive. The interval between the first and second doses canalso be from about 2 to about 4 weeks, with the preferred interval beingabout 3 weeks.

The methods also include an optional third dose administered orally Mdays following administration of the second dose. Here, M is an integerof about 7 to about 35, inclusive, but is typically an integer fromabout 14 to about 28, inclusive. The interval between the second andthird doses can also be from about 2 to about 4 weeks, with thepreferred interval being about 3 weeks. Thus, from Day 0 (day ofadministration of the first dose) M would be an integer from about 28 toabout 56, inclusive, but is typically an integer from about 35 to about49, inclusive. The interval between the first and third doses can alsobe from about 5 to about 7 weeks, with the preferred interval beingabout 6 weeks.

The methods also include an annual oral administration of a vaccinegiven about one year following administration of the first vaccine.Generally, this dose is given from about four weeks before to about fourweeks after the one year anniversary of the first vaccine. The attendingveterinarian would determine the need for and frequency of anysubsequent booster vaccines based on the animals lifestyle and risk ofexposure.

The methods may employ any vaccine that is capable of inducing an immuneresponse in a dog. The route of administration for the vaccines includessubcutaneous, oral, oronasal, and intranasal. The first vaccine isadapted to be administered orally or subcutaneously. The second, third,and annual doses are adapted to be administered orally. Any'suitabledevice may be used to administer the vaccines, including syringes,droppers, needleless injection devices, and the like. For oraladministration, a syringe fitted with a cannula may be used to place adose of the vaccine in the dog's mouth.

In addition to the above administration schedule, the methods alsoencompass an administration schedule wherein a first dose isadministered subcutaneously. A second dose is administeredsubcutaneously about N days following administration of the firstvaccine. Here, N is an integer from about 7 to about 35, inclusive, butis typically an integer from about 14 to about 28, inclusive. Theinterval between the first and second doses can also be from about 2 toabout 4 weeks, with the preferred interval being about 3 weeks. A thirddose is administered orally M days following administration of thesecond dose. Here, M is an integer of about 7 to about 35, inclusive,but is typically an integer from about 14 to about 28, inclusive. Theinterval between the second and third doses can also be from about 2 toabout 4 weeks, with the preferred interval being about 3 weeks. Anannual oral administration of a vaccine is given about one yearfollowing administration of the first vaccine. Generally, this dose isgiven from about four weeks before to about four weeks after theone-year anniversary of the first vaccine. The attending veterinarianwould determine the need for and frequency-of any subsequent boostervaccines based on the animal's lifestyle and risk of exposure. For thisadministration schedule, the first and second doses are adapted to beadministered subcutaneously. The third and annual doses are adapted tobe administered orally.

The first, second, third, and annual doses may be the same or adifferent vaccines or immunogenic composition and each comprises,independently, one or more antigens. The vaccines are based upon live orkilled viruses and killed bacterial strains. Useful vaccines thusinclude live virus vaccines, modified-live virus vaccines, andinactivated virus vaccines, either alone or in combination thereof. Liveand modified-live vaccines contain strains that do not cause disease indogs and have been isolated in non-virulent form or have been attenuatedusing methods well known in the art including serial passage in asuitable cell line or exposure to ultraviolet light or a chemicalmutagen. Inactivated or killed vaccines contain strains which have beeninactivated by known methods, including treatment with formalin,betapropriolactone (BPL), binary ethyleneimine (BEI), or other methodsknown to those skilled in the art.

The vaccines can contain antigens for immunizing dogs to protect againstor treat diseases, including but not limited to 1) canine distempercaused by canine distemper virus; 2) infectious canine hepatitis causedby canine adenovirus type 1; 3) respiratory disease caused by canineadenovirus type 2 or canine respiratory coronavirus; 4) canineparainfluenza caused by canine parainfluenza virus; 5) enteritis causedby canine coronavirus or canine parvovirus; 6) leptospirosis caused byLeptospira canicola, L. grippotyphosa, L. icterohaemorrhagiae, L.pomona, or L. Bratislava; and 7) infectious tracheobronchitis (“kennelcough”) caused by Bordetella bronchiseptica. Examples of suitablevaccines include those in the VANGUARD® product line, including but notlimited to VANGUARD® Plus 5, VANGUARD® Plus 5 L4, and VANGUARD® Plus 5L4 CV. One vaccine may be combined with other vaccines to produce apolyvalent vaccine product that can protect dogs against a wide varietyof diseases caused by other pathogens. Currently, commercialmanufacturers of canine vaccines, as well as end users, preferpolyvalent vaccine products.

For vaccines containing modified live viruses or attenuated viruses, atherapeutically effective dose generally ranges from about 10² TCID₅₀ toabout 10¹⁰ TCID₅₀, inclusive. For specific viruses, a therapeuticallyeffective dose is generally in the following ranges: for CD virus, fromabout 10² TCID₅₀ to about 10⁸ TCID₅₀, inclusive; or from about 10³TCID₅₀ to about 10⁶ TCID₅₀, inclusive; or from about 10⁴ TCID₅₀ to about10⁵ TCID₅₀, inclusive; for CAV-2, from about 10² TCID₅₀ to about 10⁸TCID₅₀, inclusive; or from about 10³ TCID₅₀ to about 10⁶ TCID₅₀,inclusive, or from about 10⁴ TCID₅₀ to about 10⁵ TCID₅₀, inclusive; forCPV, from about 10³ TCID₅₀ to about 10¹⁰ TCID₅₀, inclusive; or fromabout 10⁶ TCID₅₀ to about 10⁹ TCID₅₀, inclusive; or from about 10⁷TCID₅₀ to about 10⁸ TCID₅₀, inclusive; and for CPI virus, from about 10³TCID₅₀ to about 10¹⁰ TCID₅₀, inclusive; or from about 10⁵ TCID₅₀ toabout 10⁹ TCID₅₀, inclusive; or from 10⁶ TCID₅₀ to about 10⁸ TCID₅₀,inclusive. The amount of CCV in an inactivated viral preparation shouldbe at least about 100 relative units per dose, and preferably in therange from about 1,000 to about 4,500 relative units per dose.

For vaccines containing a bacterin, a therapeutically effective dose foreach Leptospiral species in the vaccine generally is in the range fromabout 100 nephelometric units (NU) to about 3,500 NU per vaccine dose,and preferably in the range from about 200 NU to about 2,000 NU perdose. A therapeutically effective dose for Bordetella bronchiseptica inthe vaccine generally is in the range from about 3×10⁶ to about 3×10¹¹cells inclusive; or from about 3×10⁷ to about 3×10¹⁰ cells inclusive; orfrom about 3×10⁸ to about 3×10⁹ cells inclusive.

Other components of vaccines may include pharmaceutically acceptableexcipients, including carriers, solvents, and diluents, isotonic agents,buffering agents, stabilizers,-preservatives, immunomodulatory agents(e.g., interleukins, interferons, and other cytokines),vaso-constrictive agents, antibacterial agents, antifungal agents, andthe like. Typical carriers, solvents, and diluents include water,saline, dextrose, ethanol, glycerol, and the like. Representativeisotonic agents include sodium chloride, dextrose, mannitol, sorbitol,lactose, and the like. Useful stabilizers include gelatin, albumin, andthe like.

As used herein, “a pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, adjuvants, stabilizing agents,diluents, preservatives, antibacterial and antifungal agents, isotonicagents, adsorption delaying agents, and the like. The carrier(s) must be“acceptable” in the sense of being compatible with the components of theinvention and not deleterious to the subject to be immunized. Typically,the carriers will be will be sterile and pyrogen-free, and selectedbased on the mode of administration to be used. It is well known bythose skilled in the art that the preferred formulations for thepharmaceutically accepted carrier which comprise the vaccines are thosepharmaceutical carriers approved in the applicable regulationspromulgated by the United States (US) Department of Agriculture, orequivalent government agency in a non-US country. Therefore, thepharmaceutically accepted carrier for commercial-production of thevaccines is a carrier that is already approved or will be approved bythe appropriate government agency in the US or foreign country.

The vaccine compositions optionally may include vaccine-compatiblepharmaceutically acceptable (i.e., sterile and non-toxic) liquid,semisolid, or solid diluents that serve as pharmaceutical vehicles,excipients, or media. Diluents can include water, saline, dextrose,ethanol, glycerol, and the like. Isotonic agents can include sodiumchloride, dextrose, mannitol, sorbitol, and lactose, among others.Stabilizers include albumin, among others.

The vaccines can further be mixed with one or more adjuvants that arepharmaceutically acceptable. Many such adjuvants are known in the art.Representative adjuvants include oil-based adjuvants, such as Freund'sComplete Adjuvant and Freund's Incomplete Adjuvant, mycolate-basedadjuvants (e.g., trehalose dimycolate), bacterial lipopolysaccharides,peptidoglycans (i.e., mureins, mucopeptides, or glycoproteins such asN-Opaca, muramyl dipeptide or analogs thereof), proteoglycans (e.g.,extracted from Klebsiella pneumoniae), streptococcal preparations (e.g.,OK432), BIOSTIM® (e.g., 01 K2), Iscoms (e.g., see European PatentApplication Nos. EP 109942, EP 180564 and EP 231039), aluminumhydroxide, saponin, diethylaminoethyl (DEAE)-dextran, neutral oils(e.g., miglyol), vegetable oils (e.g., arachis oil), liposomes,PLURONICO® polyols. Other adjuvants include the RIBI adjuvant system,alum, aluminum hydroxide gel, cholesterol, oil-in-water emulsions,water-in-oil emulsions, block co-polymer (CytRx, Atlanta Ga.), SAF-M(Chiron, Emeryville Calif.), lecithin in oil (e.g., AMPHIGEN® adjuvant),saponin, Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.),GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.) or othersaponin fractions, monophosphoryl lipid A, Avridine lipid-amineadjuvant, heat-labile enterotoxin from Escherichia coli (recombinant orotherwise), cholera toxin, or muramyl dipeptide, among others.

The immunogenic compositions can further include one or more otherimmunomodulatory agents such as, e.g., interleukins, interferons, orother cytokines. The immunogenic compositions can also antibiotics,including gentamicin and Merthiolate.

Dose sizes of the vaccines typically range from about one-tenth mL toabout five mL, inclusive. Typically, a dose of about one mL is used forthe oral vaccines. Each dose contains a therapeutically effective amountof the antigen or antigens that may vary depending on the age andgeneral condition of the dog, the route of administration, the nature ofthe antigen, and other factors. The amounts and concentrations of theother components of the vaccines may be adjusted to modify the physicaland chemical properties of the vaccines, and can readily be determinedby the skilled artisan. For example, adjuvants typically comprises fromabout 25 pg to about 1000 μg, inclusive, of a one-mL dose. Similarly,antibiotics typically comprise from about one μg to about 60 μg,inclusive, of a one-mL dose.

One skilled in the art can readily formulate a vaccine. The vaccines areprovided sterile and pyrogen-free. The immunogenic compositions can bemade in various forms depending upon the route of administration,storage requirements, and the like. For example, the immunogeniccompositions can be made in the form of sterile aqueous solutions ordispersions suitable for injectable use, or made in lyophilized formsusing freeze drying techniques. Lyophilized immunogenic compositions canbe reconstituted prior to use in a stabilizing solution, e.g., salineor/and HEPES, with or without adjuvant.

The subjects suitable for vaccination would include dogs that are aboutfour weeks of age or older. Thus, the first dose would be given to a dogat about four weeks of age, followed by subsequent doses according tothe timetable given herein. Certain commercially prepared vaccinesprovide directions for the timing of administration and number of dosesbased on-the potential for maternal antibody interference. For example,the product label for VANGUARD® Plus 5 L4 (Pfizer Inc) indicates thatdogs receiving the first vaccine at less than 9 weeks of age should beadministered a third orally administered one-ml dose at about threeweeks after a second dose.

Examples

The following examples are intended to be illustrative and non-limiting,and represent a few specific embodiments of the present invention.

Example 1

This study evaluated the ability of a modified live vaccine to causeseroconversion (e.g., going from a state of having no antibodies to astate of having relatively high concentrations of antibodies to specificantigens) when a vaccine was administered subcutaneously in a firstdose, then orally in a second and third dose.

Antigens of interest are Canine Adenovirus—Type 1 (CAV-1), CanineParvovirus (CPV), and Canine Distemper Virus (CDV). Thirty-three pupsbetween the ages of 6 and 9 weeks at the initiation of the study thathad no antibodies to CAV-1, CPV or CDV were randomly divided into threetreatment groups of 11 dogs each. Dogs in treatment group T01 were givenplacebo vaccine three times, three weeks apart. This treatment group wasincluded to ensure that animals were not exposed to viruses of interestin any way except through experimental vaccination. Animals in treatmentgroups T02 and T03 were given experimental vaccines first subcutaneously(on day 0), then the same vaccine orally three weeks (day 21) and sixweeks (day 42) later. The only difference between T02 and T03 was theamount of virus in the vaccines administered, with T02 receiving arelatively low dose, and T03 receiving a relatively high dose. Bloodsamples were taken weekly to measure concentrations (e.g., titers) ofspecific antibodies. Serum samples were analyzed for serum antibodiesagainst CDV, CPV, and CAV1 according to standard procedures. Presentedin Tables 1 and 2 are details of the experimental design and theInvestigational Veterinary Product (IVP).

TABLE 1 Experimental Design Vaccination Serum Group N Day 0 Day 21 Day42 Collection T01 11 SQ Oral Oral Days 0, 7, Negative saline salinesaline 14, 21, 28, Control 35, 42, 49, T02 11 SQ Oral Oral 56, and 63“low” titer “low” titer “low” titer T03 11 SQ Oral Oral “high” titer“high” titer “high” titer SQ = subcutaneous

TABLE 2 Investigational Veterinary Product (IVP) Vaccine ApproximateInput Viral Number of Volume per IVP Serial Titers per Dose (log) DosesDose CDV-CAV₂- L031006C CDV: 2.7 T01 = 0 Approx. 1 mL CPV-CPI at(diluted CAV₂: 2.2 T02 = 33 low titer 100X) CPV: 6.0 T03 = 0 CPI: 5.9Total = 33 CDV-CAV₂- L031006C CDV: 4.7 T01 = 0 Approx. 1 mL CPV-CPI atCAV₂: 4.2 T02 = 0 relatively CPV: 8.0 T03 = 33 high titer CPI: 7.9 Total= 33

All animals in T01 remained essentially free of antibodies to theantigens of interest, indicating that there was no environmentalexposure to these viruses. Animals in T02 and T03 responded similarly tothe vaccines, indicating that the dose did not significantly influencepatterns of responses. Regardless of antigen, animals given experimentalvaccine (groups T02 and T03) responded robustly to the first(subcutaneously administered) vaccine dose. For comparison andperspective, animals are considered protected from disease when theyhave antibody titers greater than or equal to 1:16 for CAV-1, 1:32 forCDV and 1:80 for CPV.

CDV-specific antibodies peaked at 28 days after the first vaccination,with mean titers of 1:3499 and 1:3668 for T02 and T03 respectively (seeFIG. 1). in the case of CDV-specific titers following subcutaneousvaccination, this concentration of antibodies may have been high enoughto interfere with the animals' abilities to subsequently react to orallyadministered vaccines. Specific antibodies against CPV followed asimilar pattern, although the peak antibody titers occurred two weeksfollowing the 1^(st) oral vaccination (mean titer of 1:5702 on day 36)for dogs in T02, and one week following the 1^(st) oral vaccination(mean titer of 1:5613 on day 28) for dogs in T03 (see FIG. 2).

Due to the differences in timing between CDV and CPV peak antibodyconcentrations, this pattern could be interpreted as an immune responseto the oral vaccination, although the initial high antibody titers againmake interpretation difficult. However, specific antibodies againstCAV-1 clearly indicate treat there is a robust immune response to oraldosing for this antigen (see FIG. 3). On the day of the first oralvaccination, day 21, animals in T02 had mean titers of CAV-1 specificantibodies of 1:5, and those in T03 had mean titers of 1:16. Two weekslater, dogs had mean antibody titers of 1:25 and 1:118 in groups T02 andT03, respectively. Even more remarkable, two weeks following the 2^(nd)orally administered vaccine, on day 56, animals had mean titers of 1:83and 1:150 in T02 and T03 respectively. Traditionally CAV-1 antibodytiters are lower than the other two antigens, which may have decreasedthe likelihood of interference caused by subcutaneous vaccination.

While high titers of antibodies specific for CDV and CPV following asubcutaneous vaccination make clear interpretation of the efficacy oforal dosing for CDV and CPV difficult, the conclusion from this study isthat dogs are capable of seroconversion in response to subcutaneousvaccination followed by oral vaccination. A secondary conclusion is thatoral vaccination is a practical and potentially effective route ofvaccine administration for dogs.

Example 2

This study evaluated the ability of a modified live vaccine to causeseroconversion (e.g., going from a state of having no antibodies to astate of having relatively high concentrations of antibodies to specificantigens) when a vaccine was administered subcutaneously or orally in afirst dose, then orally in a second and third dose.

Thirty-nine pups, between the ages of 6 and 10 weeks at the initiationof the study, and having no antibodies to Canine Adenovirus—Type 1(CAV-1), Canine Parvovirus (CPV), Canine Distemper Virus (CDV), orCanine Parainfluenza (CPI), were randomly divided into three treatmentgroups of 13 dogs each. Dogs in treatment group T01 were orally givenplacebo vaccine three times, three weeks apart. This treatment group wasincluded to ensure that animals were not exposed to viruses of interestin any way but through experimental vaccination. Animals in treatmentgroup T02 were orally given the same experimental vaccine on days 0, 21,and 42. Animals in treatment group T03 were subcutaneously givenexperimental vaccine on day 0, then the same vaccine orally three weeks(day 21) and six weeks (day 42) later. The only difference between T02and T03 was that T02 received the first dose orally, and T03 receivedthe first dose subcutaneously. Blood samples were taken weekly tomeasure concentrations (e.g., titers) of specific antibodies. Serumsamples were analyzed for serum antibodies against CDV, CPV and CAV-1according to standard procedures. (Animals vaccinated with CAV-2generate antibodies cross-reactive with CAV-1.) Animals were consideredpositive responders (i.e., protected from disease) if they had antibodytiters greater than or equal to 1:16 for CAV-1, 1:32 for CDV, and 1:16for CPV. Presented in Tables 3 and 4 are details of the experimentaldesign and the Investigational Veterinary Product (IVP).

TABLE 3 Experimental Design Vaccination Group N Day 0 Day 21 Day 42Serum Collection T01 13 Oral Oral Oral Days 0, 7, 14, 21, NegativeVehicle Vehicle Vehicle 28, 35, 42, 49, Control 56, and 63 T02 13 OralIVP Oral IVP Oral IVP T03 13 SQ IVP Oral IVP Oral IVP SQ = subcutaneousIVP = Investigational Veterinary Product

TABLE 4 Investigational Veterinary Product (IVP) Vaccine ApproximateInput Viral Number of Volume per IVP Serial Titers per Dose (log) DosesDose CDV-CAV₂- L25Aug06C CDV: 3.5 T01 = 0 Approx. 1 mL CPV-CPI at plusCDV CAV₂: 4.0 T02 = 39 high titer prep #71802 CPV: 7.1 T03 = 39 CPI: 5.2Total = 78

As presented in Table 5, all animals in the T03 group (SQ, oral, oral)seroconverted against the three measured viruses by Day 63. All animalsin the T02 group (oral, oral, oral) seroconverted against CPV and CAV-1by Day 63. Approximately half of the animals in the T02 groupseroconverted to CDV by Day 63, suggesting that changes to the currentformulation may be possible. Thus, these results demonstrate that oraldosing is an effective approach to vaccination.

TABLE 5 Seroconversion Results Seroconversion Day of Study Results 21 4263 CPV T01 0/13  0/13  0/13 T02 7/13 10/13 13/13 T03 13/13  13/13 13/13CDV T01 0/13  0/13  0/13 T02 5/13  6/13  6/13 T03 13/13  13/13 13/13CAV1 T01 5/13 11/13 10/13 T02 12/13  13/13 13/13 T03 6/13 13/13 13/13

Example 3

This notional example describes the administration to dogs of a CanineDistemper-Adenovirus Type 2-Parainfluenza-Parvovirus Modified Live VirusVaccine. A freeze-dried preparation of the vaccine is asepticallyrehydrated with sterile diluent and shaken well, according to the labelinstructions. An example of such a vaccine is VANGUARD® Plus 5 (PfizerInc). This vaccine contains attenuated strains of CD virus(approximately 10⁵ TCID₅₀), CAV-2 (approximately 10⁴ TCID₅₀), CPI virus(approximately 10⁸ TCID₅₀), and CPV (approximately 10⁸ TCID₅₀)propagated on an established canine cell line.

Healthy dogs at 4 weeks of age or older are administered a 1-ml firstdose of the reconstituted vaccine either orally or subcutaneously. Thedogs are orally given a 1-ml second dose about three weeks after thefirst vaccine, followed by a third orally administered 1-ml dose atabout three weeks after the second dose. The dogs are given an annualorally administered 1-ml booster vaccine at about one year after thefirst vaccine. The attending veterinarian determines the need for andfrequency of any subsequent booster vaccine based on the animal'slifestyle and risk of exposure.

Example 4

This notional example describes the administration to dogs of a CanineDistemper-Adenovirus Type 2-Parainfluenza-Parvovirus Modified Live VirusVaccine containing a LeptospiraCanicola-Grippotyphosa-Icterohaemorrhagiae-Pomona Bacterin. Afreeze-dried preparation of the vaccine is aseptically rehydrated withsterile diluent and shaken well, according to the Label instructions. Anexample of such a vaccine is VANGUARD® Plus 5 L4 (Pfizer Inc). Thisvaccine is a freeze-dried preparation of attenuated strains of CD virus(approximately 10⁵ TCID₅₀), CAV-2 (approximately 10⁴ TCID₅₀), CPI virus(approximately 10⁸ TCID₅₀), CPV (approximately 10⁸ TCID₅₀), andinactivated whole cultures of L. canicola, L. grippotyphosa, L.icterohaemorrhagiae, and L. pomona. An adjuvanted sterile gel is used torehydrate the freeze-dried component, which is packaged with inert gasin place of vacuum.

Healthy dogs at 4 weeks of age or older are administered a 1-ml firstdose of the reconstituted vaccine either orally or subcutaneously. Thedogs are orally given a 1-ml second dose about three weeks after thefirst. Dogs receiving the first vaccine at less than 9 weeks of age areadministered a third orally administered 1-ml dose at about three weeksafter the second dose. The dogs are given an annual orally administered1-ml booster vaccine at about one year after the first vaccine. Theattending veterinarian determines the need for and frequency of anysubsequent booster vaccines based on the animal's lifestyle and risk ofexposure.

Example 5

This notional example describes the administration to dogs of a CanineDistemper-Adenovirus Type 2-Coronavirus-Parainfluenza-Parvovirus Vaccinecontaining a LeptospiraCanicola-Grippotyphosa-Icterohaemorrhagiae-Pomona Bacterin.

A freeze-dried Canine Distemper-Adenovirus Type2-Parainfluenza-Parvovirus Modified Live Virus Vaccine containing aLeptospira Canicola-Grippotyphosa-Icterohaemorrhagiae-Pomona Bacterin(for example VANGUARD® Plus 5 L4, see Example 4) is asepticallyrehydrated with a liquid coronavirus vaccine and shaken well, accordingto the Label instructions. An example of such a coronavirus vaccine is aliquid preparation of inactivated canine coronavirus (CCV) with anadjuvant. The combination of VANGUARD® Plus 5 L4 and a CCV vaccine issold by Pfizer as VANGUARD® Plus 5 L4 CV.

Healthy dogs at 4 weeks of age or older are administered a 1-ml firstvaccine of the reconstituted vaccine either orally or subcutaneously.The dogs are orally given a 1-ml second dose of the combined vaccineabout three weeks after the first vaccine. The dogs are orally given a1-ml dose of only the coronavirus vaccine about three weeks after thesecond dose. Dogs receiving the first vaccine at less than 9 weeks ofage receive a second orally administered 1-ml dose of the combinedvaccine (in place of the coronavirus vaccine) at about three weeks afterthe second vaccine. The dogs are given an annual orally administered1-ml booster of the combined vaccine at about one year after the primingvaccine. The attending veterinarian determines the need for andfrequency of any subsequent booster vaccines based on the animal'slifestyle and risk of exposure.

Example 6

This notional example describes the administration to dogs of a CanineDistemper-Adenovirus Type 2-Parainfluenza-Parvovirus Modified Live VirusVaccine containing a Bordetella bronchiseptica Bacterin. A freeze-driedpreparation of the vaccine is aseptically rehydrated with a liquiddiluent containing inactivated whole cultures of B. bronchiseptica andshaken well, according to the Label instructions. An example of such avaccine is VANGUARD® Plus 5 (see Example 4). An example of such adiluent is a liquid preparation of an inactivated culture of B.bronchiseptica.

Healthy dogs at 4 weeks of age or older are administered a 1-ml firstvaccine of the reconstituted vaccine either orally or subcutaneously.The dogs are orally given a 1-ml second dose of the vaccine from abouttwo to about four weeks after the first vaccine. Dogs receiving thefirst vaccine at less than four months of age receive a third orallyadministered 1-ml dose of the vaccine at about four months of age. Thedogs are given an annual orally administered 1-ml booster of the vaccineat about one year after the priming vaccine. Subsequent oral 1-mlbooster vaccines are administered annually. Where B. bronchiseptica andcanine virus exposure is likely, such as breeding, boarding, and showingsituations, an additional booster may be indicated or annualrevaccination should be timed 2 to 4 weeks prior to these events.

It should be noted that, as used in this specification and the appendedclaims, singular articles such as “a,” “an,” and “the,” may refer to oneobject or to a plurality of objects unless the context clearly indicatesotherwise. Thus, for example, reference to a composition containing “acompound” may include a single compound or two or more compounds.

It is to be understood that the above examples and descriptions areintended to be illustrative and not restrictive. Many embodiments willbe apparent to those of skill in the art upon reading the abovedescription. The scope of the invention should, therefore, be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled.

All of the methods disclosed and claimed herein can be executed withoutundue experimentation in light of the present disclosure. While themethods of this invention have been described in terms of variousembodiments, it will be apparent to those of skill in the art thatvariations may be applied to methods and in the steps or in the sequenceof steps of the method described herein without departing from theconcept, spirit, and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. A method of treating a dog for canine diseases comprisingadministering to the dog therapeutically effective amounts of vaccine,wherein the vaccine comprises viral antigens, a bacterin, or both, andwherein the vaccine is administered subcutaneously or orally in a firstdose, orally in a second dose, orally in an optional third dose, andorally in one or more annual doses, and wherein the viral antigenscomprise one or more of 1) canine distemper (CD) virus, 2) canineadenovirus type 2 (CAV-2), 3) canine parainfluenza (CPI) virus, 4)canine parvovirus (CPV), 5) and canine coronavirus (CCV), and whereinthe bacterin comprises one or more bacteria selected from Leptospiracanicola, L. grippotyphosa, L. icterohaemorrhagiae, L. pomona, L.bratislava, and Bordetella bronchiseptica; and any combination of viralantigens and bacteria thereof.
 2. (canceled)
 3. A method according toclaim 1, wherein the viral antigens are CD virus, CAV-2, CPI virus, andCPV.
 4. A method according to claim 1, wherein the viral antigens are CDvirus, CAV-2, CPI virus, and CPV, and the bacteria in the bacterin areLeptospira canicola, L. grippotyphosa, L. icterohaemorrhagiae, and L.pomona.
 5. A method according to claim 1, wherein the viral antigens areCD virus, CAV-2, CPI virus, CPV, and CCV, and the bacteria in thebacterin are Leptospira canicola, L. grippotyphosa, L.icterohaemorrhagiae and L. pomona.
 6. A method according to claim 1,wherein the viral antigens are CD virus, CAV-2, CPI virus, and CPV, andthe bacterium in the bacterin is Bordetalla bronchiseptica.
 7. A methodaccording to claim 1, wherein the canine diseases comprise one or moreof 1) CD caused by CD virus; 2) infectious canine hepatitis caused byCAV-1; 3) respiratory disease caused by CAV-2 or respiratory CCV; 4) CPIcaused by CPI virus; 5) enteritis caused by CCV or CPV; 6) leptospirosiscaused by Leptospira canicola, L. grippotyphosa, L. icterohaemorrhagiae,L. pomona, or L. Bratislava; and 7) infectious tracheobronchitis(“kennel cough”) caused by Bordetella bronchiseptica.
 8. A methodaccording to claim 7, wherein the diseases comprise 1) CD caused by CDvirus; 2) infectious canine hepatitis caused by CAV-1; 3) respiratorydisease caused by CAV-2; 4) CPI caused by CPI virus; 5) and canineparvoviral enteritis caused by CPV.
 9. A method according to claim 1,wherein the viral antigens are present in the following ranges ofamounts: for CD virus, about 102 TCID₅₀ to about 10⁸ TCID₅₀, inclusive;for CAV-2, about 10² TCID₅₀ to about 10⁸ TCID₅₀, inclusive; for CPV,about 10³ TCID₅₀ to about 10¹⁰ TCID₅₀, inclusive; for CPI virus, about10³ TCID₅₀ to about 10¹⁰ TCID₅₀, inclusive; and for CCV, at least about100 relative units (RU) per dose.
 10. (canceled)
 11. (canceled)
 12. Amethod according to claim 1, wherein each Leptospira is present in arange of amounts from about 100 nephelometric units (NU) to about 3,500NU per vaccine dose, and wherein the Bordetella bronchiseptica ispresent in a range from about 3×10⁶ to about 3×10¹¹ cells inclusive. 13.(canceled)
 14. (canceled)
 15. A method according to claim 1, wherein thesecond dose is administered from 7 to 35 days, inclusive, after thefirst dose.
 16. (canceled)
 17. A method according to claim 1, whereinthe third dose is administered from 7 to 35 days, inclusive, after thesecond dose.
 18. (canceled)
 19. A method according to claim 1, wherein afirst annual dose is administered about one year after the first dose.20. A method according to claim 19, wherein annual doses administeredafter said first annual dose are administered repeatedly about one yearafter the immediately prior annual dose.
 21. A method of treating a dogfor canine diseases comprising administering to the dog therapeuticallyeffective amounts of vaccine, wherein the vaccine comprises viralantigens, a bacterin, or both, and wherein the vaccine is administeredsubcutaneously in a first and in a second dose, and orally in a thirddose, and orally in one or more annual doses, and wherein the viralantigens comprise one or more of 1) CD virus, 2) CAV-2, 3) CPI virus, 4)CPV, 5) and CCV, and the bacterin comprises one or more bacteriaselected from Leptospira canicola, L. grippotyphosa, L.icterohaemorrhagiae, L. pomona, L. bratislava, and Bordetellabronchiseptica; and any combination of viral antigens and bacteriathereof.
 22. A method according to claim 21, wherein the viral antigensare CD virus, CAV-2, CPI virus, and CPV.
 23. A method according to claim21, wherein the viral antigens are CD virus, CAV-2, CPI virus, and CPV,and the bacteria in the bacterin are Leptospira canicola, L.grippotyphosa, L. icterohaemorrhagiae, and L. pomona.
 24. A methodaccording to claim 21, wherein the viral antigens are CD virus, CAV-2,CPI virus, CPV, and CCV, and the bacteria in the bacterin are Leptospiracanicola, L. grippotyphosa, L. icterohaemorrhagiae, and L. pomona.
 25. Amethod according to claim 21, wherein the viral antigens are CD virus,CAV-2, CPI virus, and CPV, and the bacterium in the bacterin isBordetella bronchiseptica.
 26. A method according to claim 21, whereinthe canine diseases comprise one or more of 1) CD caused by CD virus; 2)infectious canine hepatitis caused by CAV-1; 3) respiratory diseasecaused by CAV-2 or respiratory CCV; 4) CPI caused by CPI virus; 5)enteritis caused by CCV or CPV; 6) leptospirosis caused by Leptospiracanicola, L. grippotyphosa, L. icterohaemorrhagiae, L. pomona, or L.Bratislava; and 7) infectious tracheobronchitis (“kennel cough”) causedby Bordetella bronshiseptica.
 27. A method according to claim 26,wherein the diseases comprise 1) CD caused by CD virus; 2) infectiouscanine hepatitis caused by CAV-1; 3) respiratory disease caused byCAV-2; 4) CPI caused by CPI virus; 5) and canine parvoviral enteritscaused by CPV.
 28. A method according to claim 21, wherein the viralantigens are present in the following ranges of amounts: for CD virus,about 10² TCID₅₀ to about 10⁸TCID₅₀, inclusive; for CAV-2, about 10²TCID₅₀ to about 10⁸ TCID₅₀, inclusive; for CPV, about 10³ TCID₅₀ toabout 10¹⁰ TCID₅₀, inclusive; for CPI virus, about 10¹⁰ TCID₅₀, to about10¹⁰ TCID₅₀, inclusive; and for CCV, at least about 100 relative units(RU) per dose.
 29. A method according to claim 21, wherein eachLeptospira is present in a range of amounts from about 100 nephelometricunits (NU) to about 3,500 NU per vaccine dose, and wherein theBordetella bronchiseptica is present in a range from about 3×10⁶ toabout 3×10¹¹ cells inclusive.
 30. A method according to claim 21,wherein the second dose is administered from 7 to 35 days, inclusive,after the first dose.
 31. A method according to claim 21, wherein thethird dose is administered from 7 to 35 days, inclusive, after thesecond dose.
 32. A method according to claim 21, wherein a first annualdose is administered about one year after the first dose.
 33. A methodaccording to claim 32, wherein annual doses administered after saidfirst annual dose are administered repeatedly about one year after theimmediately prior annual dose,